Bones provide rigid support for the body, mechanical integrity of movement and protection, and serve as a site of mineral homeostasis. Additionally, bone is an indispensable connective tissue and the primary site for hematopoiesis. Bone is continuously remodeled through new bone formation by osteoblast cells and by resorption of old bone by osteoclast cells, a renewal process which provides the skeleton with structural and functional integrity (Boyle et al., Nature 423:337-342 (2003); Teitelbaum, Science 289:1504-1508 (2000); Suda et al., Endocr. Rev. 20:345-357 (1999); Stein et al., In Principles of Bone Biology. (Bilezikian, Raisz, Rodan, eds.) Academic Press (1996)). Thus, bone remodeling occurs through the coupled actions of osteoblasts and osteoclasts.
Fresh layers of osteoid, a cement-like substance, are spread down onto existing bone by osteoblasts. Bone formation is completed when hyroxyapatite crystals are deposited on the osteoid. Osteoclasts, the primary cells responsible for bone resorption, arise from hematopoietic cells of the macrophage/monocyte lineage and are multinucleated cells (i.e., polykaryons) that form by fusion of monocytes. Osteoclasts adhere to bone and remove it by acidification and proteolytic digestion. Tunnels are then formed in the bone, and the tunnels function as pathways for osteoblasts and small blood vessels. New layers of osteoid are deposited inside the tunnels and this eventually becomes new bone matrix (Boyle et al., supra, 2003; Teitelbaum, supra, 2000; Suda et al., supra, 1999; Stein et al., supra, 1996). Bone homeostasis is, thus, also maintained by coupled actions of osteoblasts and osteoclasts.
Despite being derived from the same bone marrow precursor cells of the monocyte-macrophage lineage that give rise to macrophages and dendritic cells, osteoclasts are the only cells capable of resorbing bone (Boyle et al., supra, 2003; Teitelbaum, supra, 2000; Suda et al., supra, 1999). The differentiation of osteoclasts from myelomonocytic precursors is tightly regulated and supported by the activity of osteoblasts. Hence, many of the osteotropic factors modulating osteoclast differentiation have been identified and are shown to exert their actions by regulating osteoblasts.
Through the study of various spontaneous and induced mutant mice, there has been considerable progress made in the field of osteoclast development. Osteoclasts secrete various enzymes that act in dissolution of bone material. For example, tartrate resistant acid phosphatase (TRACP) decalcifies the bone, while cathepsin K digests the bone matrix proteins. Osteoclasts also acidify the surrounding environment through vacuolar H+-ATPase activity, thereby further promoting bone disruption. Other cell-autonomous factors for osteoclast differentiation have been identified using various knockout mice. c-Fos KO mice also fail to generate osteoclasts, and thus become osteopetrotic, but they produce macrophages (Wang et al., Nature 360:741-745 (1992); Grigoriadis et al., Science 266:443-448 (1994)). Mice lacking both p50 and p52 subunits of NF-κB display defects in osteoclast development similar to those seen in c-Fos KO mice (Iotsova et al., Nat. Med. 3:1285-1289 (1997); Franzoso et al., Genes Dev. 11:3482-3496 (1997)).
Osteoblasts induce osteoclastogenesis from bone marrow precursors, and the process is influenced by various cells producing osteotropic factors that modulate bone homeostasis. These factors can be divided into three groups: 1) those influencing the activity of osteoblasts (e.g., parathyroid hormone (PTH) or 1,25-dihydroxyvitamin DS (1,25(OH)2D3 [referred to herein as “Vit-D3”] regulating the expression of TRANCE in osteoblasts); 2) those affecting osteoclast precursors or osteoclasts per se (e.g., the putative ligands for OSCAR or TREM); and 3) those with bipotential effects (e.g., TGF-β can either inhibit or promote osteoclast differentiation by acting on osteoblasts or osteoclasts, respectively). Osteoblasts provide at least two factors required for osteoclastogenesis, TRANCE (TNF [tumor necrosis factor]-related activation-induced cytokine) and M-CSF (mononuclear phagocyte colony-stimulating factor), as well as a critical inhibitory factor, OPG (osteoprotegerin).
In addition, factors from osteoblasts have also been shown to be essential for osteoclast differentiation. The osteoblast protein RANKL (receptor for activating NF-κB ligand) also called osteoclast differentiation factor (ODF) (Yasuda et al., Proc. Natl. Acad. Sci. USA 95:3597-3602 (1998b), osteoprotegerin ligand (OPGL) (Lacey et al., Cell 93:165-176 (1998)), or TRANCE (Wong et al., J. Bio. Chem. 272:25190-25194 (1997)), is a cytokine belonging to the TNF (tumor necrosis factor) family (Anderson et al., Nature 390:175-179 (1997)). “RANK” refers to TRANCE/RANK, and “OPG” refers to OPG/OCIF/TR1/FDCR-1, (Simonet et al., Cell, 89:309-319, (1997); Yasuda et al., Endochronol. 139:1329-1337 (1998a); Tan et al., Gene 204:35-46 (1997); Kwon et al., FASEB J. 12:845-854, (1998); Yun et al., J. Immunol. 161:6113-6121 (1998), based on the chronological order of publication.) For simplicity, therefore, the term “TRANCE” refers herein to “TRANCE/RANKL/OPGL/ODF.”).
TRANCE is a key regulator that stimulates differentiation of osteoclast precursor cells and activates mature osteoclasts. Thus, it plays a major role in homeostasis of the bone by inducing differentiation (Lacey et al., supra, 1998) and the osteoblast-mediated activation of bone resorption by osteoclasts (Fuller et al., supra, 1998; Jimi et al., J. Immunol. 163:434-442 (1999). It also inhibits apoptosis of osteoclasts (Fuller et al., J. Exp. Med. 188:997-1001 (1998)). These functions of TRANCE/RANKL/OPGL/ODF are mediated by binding to its receptor, RANK (receptor activator of NF-κB). In vivo, the direct role of TRANCE in osteoclastogenesis, and osteoclast differentiation has been demonstrated by, e.g., Kim et al., Proc. Natl. Acad. Sci. USA 97:10905-10910 (2000), and Kong et al., Nature 397:315-323 (1999).
Recent findings also show that the expression of TRANCE and optimal TRANCE-induced osteoclast differentiation and bone-resorbing activity requires the action of various bone resorbing hormones (e.g., Vit-D3 or PTH) and co-stimulatory receptors (e.g., OSCAR or TREM), and additional soluble factors, such as inflammatory cytokines (e.g., IL-1, IL-6, IL-11 and TNF-α), glucocorticoids, and parathyroid hormone (PTH). Calcitonin, and prostaglandin E2 also regulate osteoclast activity (Boyle et al., supra, 2003; Teitelbaum, supra, 2000; Suda et al., supra, 1999). Moreover, that the action of TRANCE on osteoclast precursors in osteoclast differentiation is potentiated by additional cytokines and co-stimulatory factors (e.g., IL-1, TNF-β, and the putative ligands for OSCAR or TREM), and counteracted upon by inhibitory molecules (e.g., GM-CSF, INF-γ and INF-β) (Boyle et al., supra, 2003; Teitelbaum, supra, 2000; Suda et al., supra, 1999; Koga et al., Nature 428:758-763 (2004); Mocsai et al., Proc. Natl. Acad. Sci. USA 101:6158-6163 (2004); Kim et al., J. Exp. Med. 195:201-209 (2002); Cella et al., J. Exp. Med. 198:645-651 (2003); Colonna, Nat. Rev. Immunol. 3:445-453 (2003); Colonna, J. Clin. Invest. 111:313-314 (2003); Jimi et al., J. Biol. Chem. 271:4605-4608 (1996); Jimi et al., J. Biol. Chem. 273:8799-8805 (1998); Takayanagi et al., Nature 408:600-605 (2000); Fox et al., J. Immunol. 165:4957-4963 (2000); Sells Galvin et al., Biochem. Biophys. Res. Commun. 265:233-239 (1999); Massey et al., Bone 28:577-582 (2001); Takayanagi et al., Nature 416:744-749 (2002); Lee et al., Endocrinol. 144:3524-3531 (2003)).
Thus, the presence of TRANCE up-regulators leads to enhanced bone resorption and a corresponding loss of bone mass, further indicating that TRANCE, like M-CSF, is one of the factors provided by osteoblasts for osteoclast differentiation. In addition, when recombinant M-CSF and TRANCE are added to bone marrow cells or spleen cells in culture they differentiate into bone-resorbing mature osteoclasts, even in the absence of osteoblasts/stromal cells. TRANCE KO mice are osteopetrotic due to defects in osteoclast development, although TRANCE KO mice have normal macrophages.
Osteoblasts also produce a decoy ligand, osteoprotegrin (OPG), which competes with TRANCE and inhibits its activity. OPG production is up-regulated by cytokines IL-1 and TNF-α, steroid hormone β-estradiol, and mechanical stress, thereby stimulating bone formation. In contrast, gluococorticoids, PTH, and prostaglandins suppress production of OPG, while enhancing the expression of TRANCE, and thus, enhance bone resorption. Thus, the net effect of pro-osteoclastogenic factors on osteoblasts is, in general, to increase the ratio between TRANCE and OPG, maximizing the capacity of activated osteoblasts to induce osteoclast differentiation (Boyle et al., supra, 2003; Teitelbaum, supra, 2000; Suda et al., supra, 1999; Lee et al., J. Immunol. 169:2374-2380 (2002); Lee et al., Endocrinol. 140:3552-3561 (1999); Kimble et al., J. Biol. Chem. 271:28890-28897 (1999); Thirunavukkarasu et al., J. Biol. Chem. 275:25163-25172. (2000); Thirunavukkarasu et al. J. Biol. Chem. 276: 36241-36250 (2001); Halladay et al., J. Cell Biochem. 84:1-11 (2001); Kondo et al., J. Bone. Miner. Res. 19:1411-1419 (2004); Quinn et al., supra, 2001; Takai et al., supra, 1998; Brandstrom et al., Biochem. Biophys. Res. Commun. 247:338-341 (1998); Brandstrom et al., Biochem. Biophys. Res. Commun. 248:454-457 (1998); Chen et al., Horm. Metab. Res. 36:674-678 (2004); Hofbauer et al., Endocrinol. 140:4367-4370 (1999); Vidal et al., Biochem. Biophys. Res. Commun. 248:696-700 (1998); Nakamichi et al., J. Immunol. 175:1956-1964 (2005)).
Although elucidation of pro-osteoclastogenic factors produced by osteoblasts in response to bone resorbing hormones has progressed considerably in recent years, the characterization of inhibitory factors that are produced in osteoblasts, but suppressed by pro-osteoclastogenic factors has been more limited. OPG expression is suppressed in osteoblasts in response to pro-osteoclastogenic factors, although OPG is indeed a critical inhibitor that should be suppressed to promote osteoclastogenesis. Thus, it is possible that other osteoblastic inhibitors exist. For example, the anti-osteoclastogenic action of TGF-β in the osteoblast-induced osteoclast differentiation system in vitro cannot be fully reversed by anti-OPG (Takai et al., J. Bio. Chem. 273:27091-27096 (1998); Murakami et al., Biochem. Biophys. Res. Commun. 252:747-752 (1998). Moreover, even when OPG-deficient osteoblasts are used in a co-culture system, TGF-β still exerts its anti-osteoclastogenic action (Quinn et al., J. Bone Mineral Res. 16:1787-1794 (2001), suggesting that additional inhibitory factors are produced by osteoblasts.
Moreover, when mature osteoclasts attach to the bone surface, a characteristic resorption pit forms below the cell at the site of attachment of the ruffled border (Boyle et al., supra, 2003; Teitelbaum, supra, 2000; Suda et al., supra, 1999). The specialized ruffled border and sealing zone appear only in activated osteoclasts during bone resorption. Mature osteoclasts express various molecules involved in bone resorption, such as carbonic anhydrase II, integrins, H+-ATPase-type proton pump, and several proteases, including cathepsins (Boyle et al., supra, 2003; Teitelbaum, supra, 2000; Suda et al., supra, 1999). In humans, mutations in carbonic anhydrase II, H+-ATPase-type proton pump, and cathepsin K have been associated with defective bone resorption by osteoclasts, indicating that these molecules are important regulators of OC function Sly et al., Proc. Natl. Acad. Sci. USA, 80:2752-2756 (1983); Yamamoto et al., J. Clin. Invest. 91:362-367 (1993); Gelb et al., Science 273:1236-1238 (1996)).
Until the present invention, only limited progress had been made in determining the nature of such inhibitors. The question of whether other osteoblast-produced inhibitors are regulated like OPG expression in response to osteotropic factors or whether their regulation contributes to the mechanisms of bone homeostasis regulated by osteotropic factors remained unanswered. It is, however, likely that additional molecules, yet-to-be identified, are differentially produced by osteoblasts in response to various osteotropic factors, either to promote or inhibit osteoclast differentiation. In addition, expression of some factors may need to be regulated coordinately with TRANCE or OPG in osteoblasts for proper bone homeostasis. Further elucidation of such molecules that mediate the communication between osteoblasts and osteoclasts is required to fully understand how bone homeostasis is maintained, and to develop better therapeutics for various diseases in bone.
Thus, there remains a need in the art for the identifying and further characterizing additional co-stimulators and inhibitors, which is crucial for understanding how osteoclast differentiation is regulated. Since osteoclasts are the principal, if not the only, cells which can resorb bone, understanding the molecular pathways leading to the differentiation and activation of osteoclasts will improve the treatment of arthritis and degenerative bone diseases resulting in excessive bone resorption.